Interpretive Summary: Trophectoderm cell lines were established from 8-day in vitro cultured bovine embryos that were derived from fertilization (IVF), somatic cell nuclear transfer (NT), or parthenogenetic activation (P) of in vitro-matured oocytes and from five 8-day-old in vivo (V) embryos. The most abundant cellular proteins of 5 V-, 16 NT-, 12 P-, and 16 IVF-derived cell lines were compared by 2D-gel electrophoresis and mass spectrometry; that is, the unaltered thiourea/urea extract of each cell culture was analyzed. One-hundred and eighteen in common protein spots were examined, and 95% were identified with significant scores from protein and gene database searches. Of the proteins detected and identified, actin and cytokeratin-8 were found to be the most abundant. Other prominently expressed cellular proteins were metabolic enzymes such as aldose reductase, phosphoglycerate mutase, enolase, triosephosphate isomerase, cytoskeletal interacting proteins transgelin and stratifin, antioxidant proteins peroxiredoxin 1 and anti-oxidant protein 2, and the calcium-dependent lipid-binding proteins annexin I and II. In comparative analysis of the 2D-gels, the NT-derived trophectoderm showed diminished expression of annexin I and II in comparison to the IVF- and V- derived trophectoderm. Since annexins I and II are abundantly expressed in the placenta and have functions important to the maintenance of placentation, the down-regulation of the annexins in the cultured NT trophectoderm may be related to the frequent failures of NT pregnancies.

Technical Abstract:
Nuclear cloning of cattle is currently very inefficient in terms of the production and survival of nuclear cloned calves. Because of the great promise of using nuclear cloning technology in farm animals to genetically improve their production and quality traits, nuclear cloning in cattle, and other farm animals, needs to be improved. Nuclear cloning has detrimental effects on the DNA and its final “expression” in the form of the proteins that make up the body of the developing bovine fetus and of the fetus’s placenta. In fact, most problems found in nuclear cloned fetuses concern development the placenta, the organ which enables the fetus to gain nutrition from the mother. The study looked at the proteins being made by the fetus’s placental cells that specifically form the connection with the mother in the uterus. It was found that two proteins, out of the 104 assayed, were not being produced as much in cloned fetuses as in normal control fetuses made from the union of egg and sperm. These two proteins, annexin I and annexin II, could be important to placenta establishment and function in several ways, and their “down-regulation” in cloned fetuses may be associated with, or cause, the spontaneous abortions that claim 90-98% of cloned bovine fetuses. Identification of a correlation between annexin I and II down-regulation with cloning may lead to improvements in the cloning technique.